This collaborative study will be performed in Brazil at the Endocrinology Division, Medical School of the Universidade Federal do Rio Grande do Sul, as an extension of NIH grant # RO1 DK-36256 to extend studies on the function of the iodothyronine deiodinases under physiological and pathophysiological circumstances in humans. The types 1, 2, and 3 deiodinases (D1, D2, and D3) constitute a family of oxidoreductases that catalyze the removal of iodine from the outer (D1 and D2, activation) or inner ring (D1 and D3, inactivation) of the thyroid hormones. The deiodination pathway is a critical step in thyroid hormone activation and inactivation, allowing for rapid changes in intracellular thyroid status in a tissue-specific manner, without affecting systemic thyroid hormone levels. In these studies, we will explore the mechanism of catalysis in intact cells by the third member of the selenodeiodinase family, D3. The D3 enzyme catalyzes the inactivation of T4 and T3 and its overexpression, as occurs in large hemangiomas, causes severe hypothyroidism in infants and adults. Increases in D3 activity, causing local hypothyroidism, may also be important in the adaptive response of humans to illness or fasting to conserve energy. Furthermore, increased D3 activity has been demonstrated in failing hearts, indicating that reduction of local intracellular TS may contribute to a hypothyroid condition in the myocardium. Our confocal microscopic studies predict that the active catalytic center of D3 is extracellular. We will determine if thyroid hormone inactivation by D3 occurs in the extracellular space and whether catalysis can be altered by either covalent modification of the enzyme by impermeant probes or by blocking the normal intracellular cycling of D3. The studies proposed here will shed light on the function of this deiodinase and will have major implications for our understanding of factors controlling thyroid hormone levels in healthy and sick individuals. [unreadable] [unreadable] [unreadable]